Antimalarial evaluation of copper(II) nanohybrid solids: inhibition of plasmepsin II, a hemoglobin-degrading malarial aspartic protease from Plasmodium falciparum

A new class of copper(II) nanohybrid solids, LCu(CH₃COO)₂ and LCuCl₂, have been synthesized and characterized by transmission electron microscopy, dynamic light scattering, and IR spectroscopy, and have been found to be capped by a bis(benzimidazole) diamide ligand (L). The particle sizes of these nanohybrid solids were found to be in the ranges 5–10 and 60–70 nm, respectively. These nanohybrid solids were evaluated for their in vitro antimalarial activity against a chloroquine-sensitive isolate of Plasmodium falciparum (MRC 2). The interactions between these nanohybrid solids and plasmepsin II (an aspartic protease and a plausible novel target for antimalarial drug development), which is believed to be essential for hemoglobin degradation by the parasite, have been assayed by UV–vis spectroscopy and inhibition kinetics using Lineweaver–Burk plots. Our results suggest that these two compounds have antimalarial activities, and the IC₅₀ values (0.025–0.032 μg/ml) are similar to the IC₅₀ value of the standard drug chloroquine used in the bioassay. Lineweaver–Burk plots for inhibition of plasmepsin II by LCu(CH₃COO)₂ and LCuCl₂ show that the inhibition is competitive with respect to the substrate. The inhibition constants of LCu(CH₃COO)₂ and LCuCl₂ were found to be 10 and 13 μM, respectively. The IC₅₀ values for inhibition of plasmepsin II by LCu(CH₃COO)₂ and LCuCl₂ were found to be 14 and 17 μM, respectively. Copper(II) metal capped by a benzimidazole group, which resembles the histidine group of copper proteins (galactose oxidase, β-hydroxylase), could provide a suitable anchoring site on the nanosurface and thus could be useful for inhibition of target enzymes via binding to the S1/S3 pocket of the enzyme hydrophobically. Both copper(II) nanohybrid solids were found to be nontoxic against human hepatocellular carcinoma cells and were highly selective for plasmepsin II versus human cathepsin D. The pivotal mechanism of antimalarial activity of these compounds via plasmepsin II inhibition in the P. falciparum malaria parasite is demonstrated.     

Potential of lithium to reduce aluminium-induced cytotoxic effects in rat brain

The present study was aimed to explore the potential of an antidepressant drug lithium (Li) in reducing aluminium (Al) induced neurotoxicity. To carry out the investigations, Al was administered orally (100 mg AlCl₃/Kg b wt/day) whereas, Li was administered through diet (1.1 g Li₂CO₃/Kg diet, daily) for a total duration of 2 months. Al treatment resulted in a significant increase in the activity of enzyme nitric oxide synthase and the levels of l-citrulline which, however, were decreased appreciably following lithium supplementation. Al treatment also revealed an increase in DNA fragmentation as evidenced by an increase in number of comets. Interestingly, Li supplementation to Al treated rats reduced the damage inflicted on DNA by Al. Ultrastructural studies revealed an increase in chromatin condensation with discontinuity in nuclear membrane in both the cerebrum and cerebellum of Al treated rats which showed improvement following Li supplementation. Alterations in the structure of synapse and mitochondrial swelling were also seen. The present study shows the potential of Li in containing the damage inflicted by Al on rat brain.     

Activities of enzymes of fermentation pathways in the leaves and roots of contrasting cultivars of sorghum (Sorghum Bicolor L.) during flooding

Flooding evoked a differential response in the activities of enzymes of fermentation pathway in leaves and roots of flood sensitive (S-308) and flood-tolerant (SSG-59-3) cultivars of sorghum. Activities of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) and alanine aminotransferase (AlaAT) enhanced in roots of SSG-59-3 during 72 h of flooding. In contrast, a transient increase in the activities was discerned in roots of S-308 up to 24 h flooding followed by a decline in activities of these enzymes. In leaves of SSG-59-3, the activities of ADH and LDH increased to about three fold during flooding stress as compared to that in the non-flooded control plants. Though elevation in activities of these enzymes was observed in leaves of S-308 up to 48 h of flooding, the magnitude of enhancement was much lower than that in SSG-59-3. Alanine aminotranferase activity depressed in leaves of both the cultivars but the level of decline was more pronounced in sensitive cultivar S-308 as compare to tolerant SSG-59-3. The amount of alcohol, lactic acid and alanine were higher in both roots and leaves of SSG-59-3 than that in S-308 during flooding stress. It is thus apparent that roots and leaves of flood tolerant variety tends to attain greater capacity to perform reactions of various fermentation pathways to sustain production of ATP under flooded conditions.     

Concentration of a potent calcium oxalate monohydrate crystal growth inhibitor in the urine of normal persons and kidney stone patients by ELISA-based assay system employing monoclonal antibodies

Standardized calcium oxalate monohydrate (COM) crystal growth assay system was employed to study the ability of various test samples to influence growth rates of COM crystals. The inhibitory activity (IA) of various samples was expressed in terms of inhibitory units. Urine samples obtained from normal persons and kidney stone patients were found to have IA of 3.18 +/- 0.62 and 1.02 +/- 0.08, respectively. A potent inhibitor having molecular weight between 14.2 and 16.2 kDa was found to be primarily responsible for the differences observed in the urinary IAs between normal persons and kidney stone patients. The potent inhibitor was found to be tightly associated with a chromophore resembling Urobilirubin. An ELISA based assay system, using monoclonal antibodies against the above most potent inhibitor confirmed the difference observed in the urinary IA between the normal persons and kidney stone patients. This assay system has the potential to be routinely used to screen human beings for potential stone formers.     

Plant Hsp90 family with special reference to rice

Hsp90 family represents a group of highly conserved and strongly expressed proteins present in almost all biological species. Heat shock proteins in the range of 90 kDa have been detected in a range of plant species andhsp90 genes have been cloned and characterized in selected instances. However, the expression characteristics of plant Hsp90 are poorly understood. Work on expression characteristics of rice Hsp90 is reviewed in this paper. Experimental evidence is provided for indicating that while the rice 87 kDa protein is transiently synthesized within initial 2 h of heat shock, high steady-state levels of this protein are retained even under prolonged high temperature stress conditions or recovery following 4 h heat shock. It is further shown that fifteen different wild rices accumulate differential levels of these proteins in response to heat shock treatment.     

Effect of presence of a dominant follicle on the superovulatory response in buffalo (Bubalus bubalis)

Ten buffalo were superovulated by administration of 8 doses of FSH in a descending schedule spread over 4 d (5.5/5.5, 4.5/4.5, 3.5/3.5 and 2.5/2.5 mL, i.m.; total dose of 64 AU in 32 mL) beginning on Day 10 of an unstimulated estrous cycle, and 30 and 20 mg Lutalyse was given alongwith the 5th and 6th injections of FSH, respectively, to induce luteolysis. The number of corpora lutea (CL) was determined on 6 d post estrus. The ovaries were examined daily by ultrasonography from Day -5 to Day 5 (Day 0 = day of start of superovulation). The animals were retrospectively classified into 2 groups depending upon the presence (n = 4) or absence of a dominant follicle (n = 6). The mean diameter of the largest follicle (F1) increased from 8.25 +/- 0.48 mm on Day -5 to 10.75 +/- 0.25 mm on Day 0 in the dominant group, whereas in the nondominant group the F1 follicle exhibited a progressive decrease from 9.00 +/- 0.45 mm to 7.00 +/- 0.65 mm during the same period, the difference in profiles between the 2 groups was significant (P = 0.042). The profile of the diameter of the second largest follicle (F2) and the difference in diameters between largest and second largest follicles (F1-F2) were not significantly different between the 2 groups. The profile of mean number of large (> or = 10 mm diameter), but not small (2 to 5 mm diameter) or medium (6 to 9 mm diameter) follicles differed significantly (P = 0.001) between the 2 groups from Day -5 to Day 5 (P = 0.030). The number of CL was not significantly different between nondominant (4.00 +/- 0.97) and dominant groups (3.25 +/- 1.31). The number of CL was positively correlated (P < 0.01) with the number of medium follicles and the total number of follicles on the day of initiation of superovulation, but not with follicles of any size category or total number of follicles on any previous day. The results of this study indicate that following the use of morphological criteria based on the size of the largest follicle alone, the superovulation response is not affected by the presence of a dominant follicle at the initiation of superovulation in buffalo.     

On the Organisation of the Sarcotubular Systems in the Caudal Muscle Cells of Larvaceans (Tunicata)

The structure of the sarcotubular systems of the caudal muscle cells is described in various larvaceans. In Oikopleura there is both a transverse tubular system and a sarcoplasmic reticulum; there are internal couplings between the two and also sarcolemmal couplings. In Fritillaria (and probably also in Kowalevskaia), a transverse tubular system is lacking, and there are only sarcolemmal couplings with the sarcoplasmic reticulum, which is related to the mitochondria as well as to the myofilaments. The significance of these differences is discussed, and it is concluded that the arrangement of the sarcotubular systems is related to muscle fibre thickness; within the Tunicata, these systems do not indicate phylogenetic relationships.     

Fine structure of the adhesive pads of Gonionemus vertens

Summary The axonal transport of HRP in both the peripheral and central branches of dorsal root ganglion cells was studied in rats.For studying axonal transport in the peripheral branch HRP as a dry substance was applied to the peroneal nerve injured either by teasing, by cutting or crushing. After a short survival time (22 h) mainly small spinal ganglion cells of the corresponding segments were labelled, while after a prolonged survival time (70 h) mainly large cells were labelled. These labelling differences are referred to different transport rates or to differences in the process of accumulation of HRP in neurons of various sizes. No evidence could be found for HRP transport from the peripheral into the central branch.Injection of HRP into the spinal cord (survival time 22 h) or into the dorsal column nuclei (survival time 46 h) was followed by labelling of numerous spinal ganglion cell perikarya of all sizes. Reaction product was found also within the prebifurcation segment of spinal ganglion cell processes.On the basis of light microscopic exploration only somatopetal transport could be detected.This investigation was supported by the Fonds zur Förderung der wissenschaftlichen Forschung in ÖsterreichThe authors wish to thank Prof. Dr. H. Holländer and his coworkers (Neuroanatom. Abteilung, Max Planck Institut für Psychiatrie, München) for many helpful suggestions to improve the technique. Thanks are also due to Dr. E. Krammer and Dr. H. Gruber for stimulating criticism and to Miss F. Schramm for technical assistanceDedicated to Prof. H. Ferner with best wishes on his 65th birthday     

Antibody response to technetium-99m-labeled sheep erythrocytes in mice treated with anti-lymphocyte serum and concanavalin A.

In the present series of experiments we have studied the effects of anti-lymphocyte serum (ALS) and concanavlin A (Con A) on the immune response to technetium-99m-labeled sheep erythrocytes (SRBC) and have related this to the localization and persistence of antigen at the site of induction and antibody synthesis. The number of ^99mTc-labeled SRBC in the spleen and liver was quantified by gamma scintillation counting and the cellular kinetics of the splenic antibody response was determined by means of the hemolytic plaque technique. After injection of normal rabbit serum (NRS)-treated control mice with 4 × 10^899mTc-labeled SRBC, the number of cells localizing in the spleen ranged from a high of 4.2 × 10⁶ on Day 1 to a low of 1.7 × 10⁶ on Day 4, while the number in the liver ranged from a high of 68.8 × 10⁸ on Day 1 to 18.6 × 10⁶ on Day 4. The number of splenic plaque-forming cells (PFC) increased from 321–429 on Day 1 to 93,000–101,000 PFC on Day 4 and this was paralled by a rise in serum hemagglutinin and hemolysin titers. In mice treated with ALS on the other hand, splenic localization initially was increased 10-fold, hepatic localization was unchanged, and the antibody response was markedly suppressed. Splenic PFC ranged from approximately 100 between days 1 and 3 and increased to only 500 on Day 4. Mice which received Con A on Day — 1 had a reduction in splenic PFC which ranged from 150 on Day 1 to 1900 on Day 4. Splenic localization of ^99mTc-labeled RBC initially was three- to fourfold greater than that in NRS-treated mice and then decreased to control levels. The increased numbers of SRBC detected in the spleens of immunosuppressed mice at the time of peak response can be attributed to decreased in vivo lysis by reduced numbers of splenic antibody-producing cells.     

Microanatomy of the subumbrellar motor innervation inAglantha digitale (Hydromedusae: Trachylina)

Summary The hydrozoan medusaAglantha digitale (Müller 1776) has eight syncytical giant motor axons, up to 40 m in diameter, running from the margin, up the inside of the bell towards the apex. Giant motor axons injected with Lucifer Yellow CH are connected with lateral neurons running circumferentially across the subumbrellar muscle. These processes fill with the dye. Bundles of 20 to 50 small dye-coupled neurons extend circumferentially along the margin for up to 0.85mm. Giant motor axons injected with horseradish peroxidase divide into a few short branches on entering the inner nerve ring. Here the giant motor axon forms both chemical synapses and gap junctions with neurons that also send their axons into the inner nerve ring. In this region the inner and outer nerve ringe are connected by axons passing through openings in the intervening mesoglea.